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Proquest Dissertations This work is dedicated to my family. BIOCHEMICAL CHARACTERISATION OF THE EUKARYOTIC CELL CYCLE REGULATORY PROTEINS. E2F AND pRB Nadeem Ali-Khan A thesis submitted in partial fulfilment of the requirements of University College London for the degree of Doctor of Philosophy. June 2002 Division of Protein Structure, MRC National Institute for Medical Research, London. ProQuest Number: U643133 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U643133 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT Control of the cell cycle is partly mediated by a transcriptional regulatory mechanism whose components include the pRb family of tumour suppressors (pRb, p i30, p i07) and the E2F/DP heterodimeric transcription factors. Each of these heterodimers consists of one member of the E2F family of proteins (E2Fs 1- 6) and one of the DP family (DPs 1 and 2). E2F/DP activation of cell cycle genes is negatively regulated by cyclin A-CDK2-mediated phosphorylation of DP. The formation of a complex between E2F/DP and a pRb family protein leads to anti­ proliferative transcriptional repression. Binding of the Human Papillomavirus (HPV) E7 oncoprotein to pRb blocks the interaction of the tumour suppressor with E2F/DP as part of a viral cell transformation mechanism. Fragments of pRb, E2F-1 and DP-1 were over-expressed and purified by chromatographic means prior to their biochemical characterisation. Using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure, attempts were made to determine high affinity DNA-binding sites for E2F homodimers. Although such sites were not identified, important considerations relating to the SELEX protocol are highlighted by these experiments. Electrophoretic Mobility Shift Assays were used to demonstrate that the interaction of fragments of E2F-1 and DP-1 with their cognate DNA could be inhibited by phosphorylation by cyclin A-CDK2. Furthermore, mutation of one of two putative phosphorylation sites in DP-1 resulted in a reduced rate of cyclin A- CDK2-dependent loss of DNA binding. Isothermal titration calorimetry studies revealed that not only does pRb interact with the minimal pRb-binding region of E2F-1, but also with additional regions outside of the transactivation domain. I present data showing that HPV E7 competes for binding to pRb with constructs of E2F-1 incorporating these additional regions. Our results suggest that the CR3 domain of E7 competes with the marked box region of E2F-1 for binding to pRb. ACKNOWLEDGEMENTS I gratefully acknowledge my supervisor Dr. Steve Gamblin for his help, advice and support throughout the course of this study. I would also like to thank other members of the Division of Protein Structure, particularly Dr. B. Xiao, Dr. D. Emery and Dr. I. Tews for their assistance and guidance with experimental aspects of the work, as well as Dr. P. Walker for help with the computing. I am also grateful to members of the Photographic Department at the NIMR, particularly Frank, for their help with the figures presented in this thesis. To members of my family, particularly my mother, I owe an immense debt of gratitude for their unfailing support. I would also like to thank my friends, especially Anwara and Ayçe for always being there. CONTENTS ABSTRACT 3 ACKNOWLEDGEMENTS 5 CONTENTS 6 LIST OF FIGURES 11 LIST OF TABLES 13 ABBREVIATIONS 14 CHAPTER 1 INTRODUCTION 16 1.1 The Eukaryotic Cell Cycle 18 1.1.1 Checkpoints and Feedback Controls 20 1.2 Transcriptional Regulation of the Cell Cycle 22 1.2.1 Cancer and the Transcriptional Regulatory Mechanism 27 1.3 The Retinoblastoma Tumour Suppressor Gene Product (pRb) 29 1.3.1 Structural Aspects of pRb 29 1.3.2 Regulation of pRb 33 1.3.3 Mechanisms of pRb-Induced Transcriptional Repression 35 1.3.3.1 Repression of RNA polymerase Il-directed Transcription 35 1.3.3.2 pRb-induced Repression of RNA polymerases I and III 43 1.4 The E2F Family of Transcription Factors 45 1.4.1 Structural Aspects 45 1.4.2 The Regulation of E2F 48 1.4.2.1 Synthesis of E2F Transcription Factors 48 1.4.2.2 Pocket Proteins and C/EBPa 49 1.4.2.3 Phosphorylation of E2F 50 1.4.2.4 Degradation and Stabilisation of E2F 51 1.4.2.5 Subcellular Localisation of E2F 52 1.4.2.6 Acétylation of E2F 52 1.4.3 Functions of E2F 53 1.4.3.1 E2F’s Target Genes 5 3 1.4.3.2 E2F as an Activator and Repressor of Transcription 55 1.4.3.3 Specificity of E2F Family Members 58 1.4.3.4 Oncogenic, Tumour Suppressive and Apoptotic Functions ofE 2F 58 1.4.4 Mechanism of E2F Activity 59 1.4.4.1 Transcriptional Activation 59 1.4.4.2 Transcriptional Repression 61 1.4.4.3 Promoter Specificity 62 1.4.4.4 Apoptosis 63 1.5 Inactivation of pRb by Viral Oncoproteins 65 1.5.1 Structure 66 1.5.2 pRb-inactivating Function 68 1.5.3 Mechanism of pRb-inactivation 69 1.6 Presentation of Experimental Work 71 CHAPTER 2 METHODS AND MATERIALS 72 2.1 General 73 2.1.1 Media for Bacterial Cell Growth 73 2.1.2 Cloning 74 2.1.2.1 Restriction Enzyme Digestions 7 4 2.1.2.2 Ligation 75 2.1.2.3 Transformation 76 2.1.2.4 Selection and Purification of Recombinant Plasmids 7 6 2.1.3 Protein Analysis 77 2.1.3.1 Polyacrylamide Gel Electrophoresis (PAGE) 77 2.1.3.2 Determination of Protein Concentration 77 2.1.3.3 Electrospray Mass Spectrometry 77 2.1.4 DNA Analysis and Purification 79 2.1.4.1 Agarose Gel Electrophoresis 79 2.1.4.2 Nondenaturing Polyacrylamide Gel Electrophoresis 79 2.1.4.3 Phenol/chloroform Extraction 80 2.1.4.4 Ethanol Precipitation 80 2.1.4.5 Determination of DNA Concentration 81 2.2 Identification of Aptamers for the E2F-1 Homodimer (Methods and Materials) 82 2.2.1 Systematic Evolution of Ligands by Exponential Enrichment (SELEX) 82 2.2.1.1 Preparation of Double-Stranded Degenerate Oligonucleotide (degOLIGO) 83 2.2.2 SELEX Method 1 83 2.2.2.1 Selection and Amplification 83 22.2.2 Cloning and Sequencing 85 2.2.2.5 EMSA (Electrophoretic Mobility Shift Assay) 85 2.2.3 SELEX Method 2 86 2.2.3.1 Preparation of Control Oligonucleotide (conOLIGO) 86 2.2.3.2 Selection and Amplification 87 2.3 Regulation of E2F/DP DNA-Binding By Cyclin-CDK Dependent Phosphorylation (Methods and Materials) 89 2.3.1 Purification of E2F-1 eye 89 2.3.1.1 Strategy 89 2.3.1.2 Protocol 89 2.3.2 Purification of DP-lggg and DP-lAs 9 s 91 2.3.2.1 Strategy 91 2.3.2.2 Protocol 91 2.3.3 Electrophoretic Mobility Shift Assays (EMSAs) 93 2.3.3.1 Probe 93 2.3.3.2 Assay 9 4 2.4 Investigation of the Interactions Between pRb, E2F and HPV E7 (Methods and Materials) 97 2.4.1 Purification of pRbAB 97 2.4.1.1 Strategy 97 2.4.1.2 Protocol 98 2.4.2 Isothermal Titration Calorimetry (TTC) 100 2.4.2.1 General Points 100 2.4.2.2 Titrations 102 CHAPTER 3 IDENTIFICATION OF APTAMERS FOR THE E2F-1 HOMODIMER 104 3.1 Outline of the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) 105 3.1.1 General Procedure 105 3.1.2 Isolation of Protein-Aptamer Complexes 107 3.2 Introduction to Experiments 110 3.3 Results 113 3.3.1 Method 1 113 3.3.2 Method 2 118 3.4 Discussion 122 3.4.1 SELEX Method 1 122 3.4.2 SELEX Method 2 124 3.4.3 General Points Concerning SELEX 126 3.4.4 Crystal Structure of the E2F/DP-DNA Complex 127 3.4.5 SELEX and E2F - The Next Step 136 CHAPTER 4 REGULATION OF E2F/DP DNA-BINDING BY CYCLIN-CDK DEPENDENT PHOSPHORYLATION 138 4.1 Outline of the Electrophoretic Mobility Shift Assay (EMSA) 139 4.1.1 Principle of the EMSA 139 4.2 Introduction to Experiments 142 4.3 Results 150 4.3.1 Purification of E2F-1 eye 150 4.3.2 Purification of DP-ls98 and DP-1 As98 153 4.3.3 Phosphorylation of the E2F Transcription Factor by Cyclin A-CDK2 158 4.4 Discussion 164 CHAPTER 5 INVESTIGATION OF THE INTERACTIONS BETWEEN pRb, E2FANDHPVE7 169 5.1 Outline of Isothermal Titration Calorimetry (ITC) 170 5.1.1 The ITC Apparatus and Mode of Operation 170 5.1.2 Determination of Thermodynamic Binding Parameters 173 5.1.3 The Thermodynamic Parameters and How Interactions Influence Them 174 5.2 Introduction to Experiments 181 5.3 Results 187 5.3.1 Purification of pRbAB 187 5.3.2 Isothermal Titration Calorimetry Experiments 191 5.4 Discussion 208 5.4.1 Determination of the Minimal Fragments of pRb and E2F-1 Required for Binding 208 5.4.2 Investigation of the Specificity of the E2F/pRb Interaction 211 5.4.2.1 Crystal Structure of pRb1 (409 .426) Complex 212 5.4.2.2 Structural Role of Non-Conserved Residues Between E 2 F - 1 (409-426) &nd E2F-5(323-340) 2 1 7 5.4.3 Investigation of HPV E7-mediated Inhibition of pRb-E2F Complex Formation 219 5.4.4 Conclusions in Terms of AH° and AS° 222 CHAPTER 6 CONCLUSION 227 REFERENCES 235 10 LIST OF FIGURES Figure 1 The Eukaryotic Cell Cycle 19 Figure 2 Functional Regions of the Retinoblastoma Tumour Suppressor Gene Product (pRb) 30 Figure 3 Elements of Eukaryotic Transcriptional Initiation for Genes Transcribed by RNA Polymerase II 37 Figure 4 Proposed Mechanisms for Repression of RNA Polymerase Il-directed Transcription by pRb 38 Figure 5 Functional Regions of the Mammalian E2F and DP Proteins 47 Figure 6 Model for E2F-Mediated Transcriptional Regulation 57 Figure 7 Structural Organisation
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